Highlights
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Processes responsible for the degradation of resin-bonded interfaces are described.
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Durability of the resin–dentin bond strength may be improved by inhibiting intrinsic collagenolytic activity.
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Cross-linking agents may increase the durability of resin–dentin bonds by increasing the mechanical properties of the collagen matrix.
Abstract
Objective
Success in adhesive dentistry means long lasting restorations. However, there is substantial evidence that this ideal objective is not always achieved. Current research in this field aims at increasing the durability of resin–dentin bonds. The objective of this paper is to examine the fundamental processes responsible for the aging mechanisms involved in the degradation of resin-bonded interfaces and the potential approaches to prevent and counteract this degradation.
Methods
PubMed searches on the hybrid layer degradation were carried out. Keywords were chosen to assess hybrid layer degradation for providing up-dated information on the basis of scientific coherence with the research objective. Approaches to prevent and counteract this degradation were also reviewed.
Results
148 peer-review articles in the English language between 1982 and 2015 were reviewed. Literature shows that resin–dentin bond degradation is a complex process, involving the hydrolysis of both the resin and the collagen fibril phases contained within the hybrid layer. Collagen fibers become vulnerable to mechanical and hydraulic fatigue, as well as degradation by host-derived proteases with collagenolytic activity (matrix metalloproteinases and cysteine cathepsins). Inhibition of the collagenolytic activity and the use of cross-linking agents are the two main strategies to increase the resistance of the hybrid layer to enzymatic degradation.
Significance
This review analyzes the issues regarding the durability of the adhesive interface, and the techniques to create stable resin–dentin bonds able to resist the collagenolytic hydrolysis that are currently studied.
1
Introduction
Resin–dentin bonding involves a series of topical treatments to dentin that completely changes its physical and chemical properties from being a hydrophilic, crystalline, relatively impermeable acid-labile surface, to one that is more hydrophobic, organic, highly permeable, acid-resistant surface.
When dentin is cut or ground with high-speed drills, a 1–2 μm thick layer of cutting debris is left on the surface, called the smear layer . When the smear layer is removed by acid-etching with 37 wt% phosphoric acid for 15 s, all of the mineral phase of the smear layer and underlying 5 μm of mineralized dentin are solubilized, causing the smear layer to dissolve and exposing the underlying type I collagen fibril meshwork of the dentin matrix.
The space between the collagen fibrils (interfibrillar space) is about 30 ± 11 nm . These spaces serve as diffusion channels for solvated adhesive comonomers to infiltrate around collagen fibrils toward the base of the 5 μm deep, demineralized layer of the collagen matrix ( Fig. 1 ) forming the so-called hybrid layer ( Fig. 2 ).
Unlike typical tissue engineering applications where a synthetic scaffold is designed to be resorbed and replaced by the host’s own tissues with 2–3 months, dentin adhesion relies on in situ tissue engineering, which is designed to create a resin-enveloped collagen scaffold that, ideally, will remain in place for decades. Nevertheless, the hybrid layer may be degraded over time, leading to failure of the adhesive interface . Although the incorporation of hydrophilic and acidic resin monomers has substantially improved the initial bonding of contemporary etch-and-rinse and self-etch adhesives to intrinsically wet dental substrates, potential problems associated with these hydrophilic formulations have been reported in several in vitro and in vivo studies . The failure of the adhesive layer leads to the formation of microgaps that are readily penetrated by pathogens. Bonding failure in the presence of bacteria, esterases , and dental plaque biofilm provokes a cascade of events leading to deterioration of the adhesive interface and failure of the composite restoration. Recent studies demonstrated that dental composite restorations continue to show limited clinical service as a result of decay and fracture and recurrent decay at the composite-tooth interface has consistently been the primary reason for replacement of composite restorations .
We are entering a new era in adhesive dentistry, where resin-bonding to enamel and dentin is beginning to be understood at the nanoscopic level. The long-term success and durability of resin–dentin bonds depends upon the ability of dentists to capitalize on new discoveries being made in adhesive technology.
The objective of this paper is to examine the fundamental processes responsible for the aging mechanisms involved in the degradation of resin-bonded interfaces and the potential approaches to prevent and counteract this degradation. For this purpose, an electronic search was conducted of the PubMed database with different combination of the following search terms: collagen, dentin, hybrid layer degradation, bonding agent, adhesive system, metalloproteinases, cysteine cathepsins, cross-linking agent, EDC. The search was restricted to articles written in English related to dentin collagen degradation. Only articles published in peer-reviewed journals were included. The PubMed search included literature reviews, in vitro and in vivo studies. Articles written in other languages, without available abstracts, those related to other field were excluded. 148 peer-review articles in the English language between 1982 and 2015 were reviewed.
2
Degradation of the adhesive interface
2.1
Degradation of the adhesive resin
Chronic deterioration of the hybrid layer involves hydrolysis and leaching of the adhesive resin that has infiltrated the demineralized dentin matrix . Leaching is facilitated by water penetration into the loosely cross-linked or hydrophilic domains of the adhesive. The hydrophilic domains exhibit limited monomer/polymer conversion because of adhesive phase separation and lack of compatibility between the hydrophobic photoinitiator and the hydrophilic phase . The poorly polymerized hydrophilic phase degrades rapidly in the aqueous environment. Resin elution continues to occur while water movement along the length of the hybrid layer becomes more rapid via transport pathways form relatively large water-filled channels . The previously resin-infiltrated collagen matrix is exposed and becomes vulnerable to the attack by proteolytic enzymes . The structure of methacrylate adhesives, presenting hydrolytically susceptible groups, such as ester and urethane, as well as hydroxyl, carboxyl, and phosphate groups , may be hydrolyzed by chemical and enzymatic degradation in the oral environment .
On prolonged exposure of the restoration to oral fluids, water begins to penetrate the resin. Water initially enters the matrix by diffusion into loosely cross-linked or hydrophilic domains or may be trapped within the matrix during photopolymerization . This water plasticizes the polymer chains by acting as a molecular lubricant. Mechanical wear of the exposed adhesive may further accelerate matrix degradation by abrading the surface, increasing the surface area and allowing greater entrance of both water and enzymes. The presence of water promotes the chemical hydrolysis of ester bonds in methacrylate materials. After exposure to salivary fluids, local domains of the methacrylate network may become sufficiently degraded and/or hydrophilic to permit access of esterases, which greatly accelerate ester bond hydrolysis.
Hydrolysis is considered a primary reason for resin degradation within the hybrid layer, contributing to the reduction of bond strength over time.
2.2
Nanoleakage
Many studies have shown that small ions or molecules can diffuse into the hybrid layer in absence of detectable interfacial gap formation. Sano et al. defined nanoleakage as the presence of microporous zones beneath or within the hybrid layer that permitted tracer penetration to occur in the absence of interfacial gaps. Nanoleakage is created by the discrepancy between dentin demineralization and adhesive infiltration that occurs with total-etching adhesive systems, in the absence of evident marginal gap formation along the resin–dentin interface .
Self-etch procedures use acidic adhesive resin monomers to simultaneously demineralize and infiltrate the bonding substrate, theoretically avoiding incomplete infiltration. Tay et al. , however, reported variable degrees of silver uptake following the use of self-etching adhesives. Thus, nanoleakage is not necessarily caused by disparities between demineralization and resin-infiltration depths. Simplified one-step self-etching adhesives are highly hydrophilic and form hybrid layers that have been found to behave as permeable membranes after polymerization, permitting water movement throughout the bonded interface . Moreover, the retention of residual water in etched dentin and/or adhesives may result in regions of incomplete polymerization or increased permeability within adhesive resin matrices .
Tay et al. used ammoniacal silver nitrate to trace the distribution of absorbed water within the hybrid layer, and observed reticular and spotted patterns of silver tracer deposition as an indicator of nanoleakage expression. In the reticular mode, silver deposits were oriented perpendicular to the hybrid layer surface. The spotted mode of nanoleakage is thought to represent microdomains in the resin matrices that contain primarily hydrophilic and/or acidic functional groups, in contrast to adjacent, relatively hydrophobic, domains . The same author reported that silver uptake likely indicated areas of increased permeability within the resin matrix. Water had been incompletely removed in these zones, resulting in regions of incomplete polymerization and/or hydrogel formation of the HEMA in the adhesive.
An immunohistochemical approach that distinguishes native collagen fibrils from resin-embedded fibrils has confirmed the interpretations of nanoleakage that implicate hybrid layer porosity. This study found that collagen fibrils within the hybrid layer were not fully embedded by dentin adhesives. Thus, etch-and-rinse and self-etch adhesive systems both fail to fully infiltrate the collagen network, achieving different degrees of infiltration from the top to the bottom of the hybrid layer.
2.3
Degradation of the collagen fibrils
Degradation may also affect the collagen matrix of the hybrid layer. Complete coverage of the nanoscale irregularities on the collagen fibrils surface via passive monomer penetration may be difficult to achieve. Thus, fibrils that remain unprotected by hydrophobic resin coatings may be vulnerable to degradation and water is claimed to be a major cause of collagen degradation. Two degradation patterns have been observed within the hybrid layer: loss of resin from interfibrillar spaces and disorganization of collagen fibrils . The degree of collagen fibril envelopment varies, depending on the type of bonding agent that could lead to incompletely infiltrated zones containing denuded collagen fibrils along the bottom of the hybrid layer .
Collagen fibrils in the hybrid layer that have been incompletely encapsulated by resin monomer can be identified immune-histochemically . Advances in reagent purification and the production of highly specific monoclonal antibodies have permitted the creation of reproducible and selective immune-labeling protocols to analyze collagen or proteoglycans . These protocols use secondary antibodies that are conjugated with gold nanoparticles of different size. Breschi et al. showed that the hybrid layer created by etch-and-rinse adhesives was characterized by different degrees of resin–collagen fibril interactions. More recently Mazzoni et al. , using in situ zymography with an ELISA assay for interrogating the functional activity of dentin proteases, quantified matrix metalloproteinases (MMPs) activity within the hybrid layer created by an etch-and-rinse adhesive, showing intense and precise fluorescent localization.
2.4
Intrinsic collagenolytic activity of mineralized dentin
Recent studies have examined the contribution of host-derived proteinases to the breakdown of collagen matrices in the pathogenesis of dentinal caries and periodontal disease . These findings had important implications for dental bonding, but the first evidence of an intrinsic collagenolytic activity of host-derived matrix metalloproteinases (MMPs) in human non carious mineralized dentin came when Pashley et al. demonstrated that dentin demineralized by acid etching slowly degrades in absence of bacteria. These authors speculated that such proteolytic activity could be exerted by dentinal matrix metalloproteinases (MMPs), which had already been shown to be potentially expressed in the dentin-pulp complex .
2.5
Matrix metalloproteinases
MMPs are a class of zinc- and calcium-dependent endopeptidases. These endogenous enzymes are important components in many biological and pathological processes because of their ability to degrade almost all extracellular matrix components . Within the oral environment, considerable interest has been devoted to the detection, distribution, and function of host-derived MMPs .
Several MMPs have been identified within the dentin-pulp complex compartments. Dentin matrix has been shown to contain at least four MMPs: stromelysin-1 (MMP-3) , collagenase (MMP-8) , and gelatinases A and B (MMP-2 and MMP-9, respectively) .
Description of the involvement of MMPs in the degradation of demineralized dentin matrix was first mentioned in a study by Tjäderhane et al. . Human MMP-2, MMP-8, and MMP-9 were identified in demineralized dentinal lesions. The experiments conducted in the mentioned study provided critical evidence that bacterial acids are required for the removal of minerals in tooth decay and for the subsequent activation of host MMPs, but bacteria alone could not cause dentin matrix degradation. Therefore, it was assumed that after demineralization, activated host-MMPs would ultimately be responsible for destroying the dentin matrix in caries lesion progression . As previously mentioned, more recently Pashley et al. began discussing the likely involvement of MMPs in the degradation of poorly resin-infiltrated hybrid layers. Today, it is thought that the exposure and subsequent activation of these endogenous enzymes during dentin bonding procedures are responsible for the almost complete disappearance of portions of hybrid layers from resin–dentin bonds that were aged in water .
2.6
Cysteine cathepsins
More recently, another group of proteases were identified in compartments of both sound and carious human dentin, cysteine proteases (CPs) . Human CPs are best known from the ubiquitously expressed lysosomal cathepsins B, H, and L, and dipeptidyl peptidase I, which until recently were thought to mediate primarily housekeeping functions in the cell . Of 15 CPs genes detected, 10 were expressed in native pulp tissue and 11 in odontoblasts.
In studies by Tersariol et al. and Nascimento et al. , the potential correlation between the MMPs and cysteine cathepsin activities in intact or carious dentin, respectively, was also investigated. Results showed that MMPs and cysteine cathepsin activities expressed highly significant correlations between intact and carious dentin, even though the activities in carious lesions were approximately 10 times higher than in intact dentin. These data indicate that the collagenolytic/gelatinolytic activity of dentin may be due not only to the presence of MMPs but also to cysteine cathepsin synergic activities.
2
Degradation of the adhesive interface
2.1
Degradation of the adhesive resin
Chronic deterioration of the hybrid layer involves hydrolysis and leaching of the adhesive resin that has infiltrated the demineralized dentin matrix . Leaching is facilitated by water penetration into the loosely cross-linked or hydrophilic domains of the adhesive. The hydrophilic domains exhibit limited monomer/polymer conversion because of adhesive phase separation and lack of compatibility between the hydrophobic photoinitiator and the hydrophilic phase . The poorly polymerized hydrophilic phase degrades rapidly in the aqueous environment. Resin elution continues to occur while water movement along the length of the hybrid layer becomes more rapid via transport pathways form relatively large water-filled channels . The previously resin-infiltrated collagen matrix is exposed and becomes vulnerable to the attack by proteolytic enzymes . The structure of methacrylate adhesives, presenting hydrolytically susceptible groups, such as ester and urethane, as well as hydroxyl, carboxyl, and phosphate groups , may be hydrolyzed by chemical and enzymatic degradation in the oral environment .
On prolonged exposure of the restoration to oral fluids, water begins to penetrate the resin. Water initially enters the matrix by diffusion into loosely cross-linked or hydrophilic domains or may be trapped within the matrix during photopolymerization . This water plasticizes the polymer chains by acting as a molecular lubricant. Mechanical wear of the exposed adhesive may further accelerate matrix degradation by abrading the surface, increasing the surface area and allowing greater entrance of both water and enzymes. The presence of water promotes the chemical hydrolysis of ester bonds in methacrylate materials. After exposure to salivary fluids, local domains of the methacrylate network may become sufficiently degraded and/or hydrophilic to permit access of esterases, which greatly accelerate ester bond hydrolysis.
Hydrolysis is considered a primary reason for resin degradation within the hybrid layer, contributing to the reduction of bond strength over time.
2.2
Nanoleakage
Many studies have shown that small ions or molecules can diffuse into the hybrid layer in absence of detectable interfacial gap formation. Sano et al. defined nanoleakage as the presence of microporous zones beneath or within the hybrid layer that permitted tracer penetration to occur in the absence of interfacial gaps. Nanoleakage is created by the discrepancy between dentin demineralization and adhesive infiltration that occurs with total-etching adhesive systems, in the absence of evident marginal gap formation along the resin–dentin interface .
Self-etch procedures use acidic adhesive resin monomers to simultaneously demineralize and infiltrate the bonding substrate, theoretically avoiding incomplete infiltration. Tay et al. , however, reported variable degrees of silver uptake following the use of self-etching adhesives. Thus, nanoleakage is not necessarily caused by disparities between demineralization and resin-infiltration depths. Simplified one-step self-etching adhesives are highly hydrophilic and form hybrid layers that have been found to behave as permeable membranes after polymerization, permitting water movement throughout the bonded interface . Moreover, the retention of residual water in etched dentin and/or adhesives may result in regions of incomplete polymerization or increased permeability within adhesive resin matrices .
Tay et al. used ammoniacal silver nitrate to trace the distribution of absorbed water within the hybrid layer, and observed reticular and spotted patterns of silver tracer deposition as an indicator of nanoleakage expression. In the reticular mode, silver deposits were oriented perpendicular to the hybrid layer surface. The spotted mode of nanoleakage is thought to represent microdomains in the resin matrices that contain primarily hydrophilic and/or acidic functional groups, in contrast to adjacent, relatively hydrophobic, domains . The same author reported that silver uptake likely indicated areas of increased permeability within the resin matrix. Water had been incompletely removed in these zones, resulting in regions of incomplete polymerization and/or hydrogel formation of the HEMA in the adhesive.
An immunohistochemical approach that distinguishes native collagen fibrils from resin-embedded fibrils has confirmed the interpretations of nanoleakage that implicate hybrid layer porosity. This study found that collagen fibrils within the hybrid layer were not fully embedded by dentin adhesives. Thus, etch-and-rinse and self-etch adhesive systems both fail to fully infiltrate the collagen network, achieving different degrees of infiltration from the top to the bottom of the hybrid layer.
2.3
Degradation of the collagen fibrils
Degradation may also affect the collagen matrix of the hybrid layer. Complete coverage of the nanoscale irregularities on the collagen fibrils surface via passive monomer penetration may be difficult to achieve. Thus, fibrils that remain unprotected by hydrophobic resin coatings may be vulnerable to degradation and water is claimed to be a major cause of collagen degradation. Two degradation patterns have been observed within the hybrid layer: loss of resin from interfibrillar spaces and disorganization of collagen fibrils . The degree of collagen fibril envelopment varies, depending on the type of bonding agent that could lead to incompletely infiltrated zones containing denuded collagen fibrils along the bottom of the hybrid layer .
Collagen fibrils in the hybrid layer that have been incompletely encapsulated by resin monomer can be identified immune-histochemically . Advances in reagent purification and the production of highly specific monoclonal antibodies have permitted the creation of reproducible and selective immune-labeling protocols to analyze collagen or proteoglycans . These protocols use secondary antibodies that are conjugated with gold nanoparticles of different size. Breschi et al. showed that the hybrid layer created by etch-and-rinse adhesives was characterized by different degrees of resin–collagen fibril interactions. More recently Mazzoni et al. , using in situ zymography with an ELISA assay for interrogating the functional activity of dentin proteases, quantified matrix metalloproteinases (MMPs) activity within the hybrid layer created by an etch-and-rinse adhesive, showing intense and precise fluorescent localization.
2.4
Intrinsic collagenolytic activity of mineralized dentin
Recent studies have examined the contribution of host-derived proteinases to the breakdown of collagen matrices in the pathogenesis of dentinal caries and periodontal disease . These findings had important implications for dental bonding, but the first evidence of an intrinsic collagenolytic activity of host-derived matrix metalloproteinases (MMPs) in human non carious mineralized dentin came when Pashley et al. demonstrated that dentin demineralized by acid etching slowly degrades in absence of bacteria. These authors speculated that such proteolytic activity could be exerted by dentinal matrix metalloproteinases (MMPs), which had already been shown to be potentially expressed in the dentin-pulp complex .
2.5
Matrix metalloproteinases
MMPs are a class of zinc- and calcium-dependent endopeptidases. These endogenous enzymes are important components in many biological and pathological processes because of their ability to degrade almost all extracellular matrix components . Within the oral environment, considerable interest has been devoted to the detection, distribution, and function of host-derived MMPs .
Several MMPs have been identified within the dentin-pulp complex compartments. Dentin matrix has been shown to contain at least four MMPs: stromelysin-1 (MMP-3) , collagenase (MMP-8) , and gelatinases A and B (MMP-2 and MMP-9, respectively) .
Description of the involvement of MMPs in the degradation of demineralized dentin matrix was first mentioned in a study by Tjäderhane et al. . Human MMP-2, MMP-8, and MMP-9 were identified in demineralized dentinal lesions. The experiments conducted in the mentioned study provided critical evidence that bacterial acids are required for the removal of minerals in tooth decay and for the subsequent activation of host MMPs, but bacteria alone could not cause dentin matrix degradation. Therefore, it was assumed that after demineralization, activated host-MMPs would ultimately be responsible for destroying the dentin matrix in caries lesion progression . As previously mentioned, more recently Pashley et al. began discussing the likely involvement of MMPs in the degradation of poorly resin-infiltrated hybrid layers. Today, it is thought that the exposure and subsequent activation of these endogenous enzymes during dentin bonding procedures are responsible for the almost complete disappearance of portions of hybrid layers from resin–dentin bonds that were aged in water .
2.6
Cysteine cathepsins
More recently, another group of proteases were identified in compartments of both sound and carious human dentin, cysteine proteases (CPs) . Human CPs are best known from the ubiquitously expressed lysosomal cathepsins B, H, and L, and dipeptidyl peptidase I, which until recently were thought to mediate primarily housekeeping functions in the cell . Of 15 CPs genes detected, 10 were expressed in native pulp tissue and 11 in odontoblasts.
In studies by Tersariol et al. and Nascimento et al. , the potential correlation between the MMPs and cysteine cathepsin activities in intact or carious dentin, respectively, was also investigated. Results showed that MMPs and cysteine cathepsin activities expressed highly significant correlations between intact and carious dentin, even though the activities in carious lesions were approximately 10 times higher than in intact dentin. These data indicate that the collagenolytic/gelatinolytic activity of dentin may be due not only to the presence of MMPs but also to cysteine cathepsin synergic activities.
3
Strategies to reduce the intrinsic collagenolytic activity
As previously described, the durability of dentin bonding systems is affected by the degradation of the resin components occurring via hydrolysis of suboptimally polymerized hydrophilic resins and degradation of collagen matrices by MMPs and cysteine cathepsins . MMPs and cathepsins have been shown to be present in dentin and they seem to be responsible for the slow hydrolysis of the collagen fibrils in the hybrid layer that anchors resin composites to the underlying mineralized dentin . To prolong the durability of the resin–dentin bond, inhibition of these proteases has been recommended through the use of synthetic MMPs inhibitors such as chlorhexidine , quaternary ammonium methacrylates, or benzalkonium chloride . Alternatively, the use of collagen-cross-linkers should inactivate these enzymes to retain hybrid layer integrity and strong dentin bonding . Moreover, other approaches have been proposed to reduce the hybrid layer degradation, including dentin remineralization and ethanol wet-bonding .
3.1
Protease inhibitors
3.1.1
Chlorhexidine
Most of the experiments designed to improve the durability of dentin bonds through enzyme inhibition have been performed with the use of chlorhexidine (CHX), a potent antimicrobial agent. CHX effectively inhibits MMP-2, -8 and -9 , and cysteine cathepsins . In 2004, Pashley et al. showed convincing evidence of its efficacy in inhibiting dentin collagenolytic enzymes. Since then, several studies have demonstrated that CHX can preserve the structural integrity of hybrid layer collagen matrix and reduce time-dependent reduction in dentin bond strength both in vivo and in vitro .
Even though CHX binding rate to mineralized dentin is almost 80% lower that to demineralized dentin , low concentrations of CHX (0.05–0.2%) are sufficient to completely inhibit the collagenolytic activity of untreated dentin powder , while 0.5–2.0% concentrations are able to only partially inhibit the activity induced with acidic SE primer . Since dentin and MMPs require calcium to maintain their tertiary structure and zinc ions for their catalytic activity and CHX loses its MMP inhibition in the presence of calcium chloride , CHX-related MMP inhibition may be due to its chelating property and calcium ions released by the primer may be responsible for the loss of inhibition by CHX. This is supported by the finding that treating dentin powder with Clearfil SE Bond primer for 2 min instead of 20 s not only increased the collagenolytic activity, but also caused the loss of inhibition by 0.5% and 1.0% CHX, with only 2.0% CHX showing significant inhibition . Also the recent reports of chlorhexidine binding by dentin collagen suggest that collagen may compete with MMPs for CHX binding, requiring the use of relatively high CHX concentrations.
Incorporation of CHX at reasonably low (0.2–2.0%) concentration into methacrylate comonomers has no effect on water sorption, cause a slight decrease in conversion rate, but may even increase flexural strength and modulus of elasticity of dental adhesives . Chlorhexidine release from the polymerized adhesive is concentration-dependent and continues at a slow steady-state level . Since the idea behind the incorporation of inhibitors into adhesive is their continuous release to prevent collagen degradation for a prolonged time, these results are promising ( Fig. 3 ).
3.1.2
Galardin
Galardin is a synthetic MMP inhibitor that exhibits strong activity against MMP-1, -2, -8, and -9. It has a collagen-like structure that binds to the active sites of MMPs. The inhibitory effect of galardin on dentinal MMPs has been confirmed by zymography and interfacial nanoleakage expression after 1 year . Moreover, its incorporation in a commercial primer resulted in inhibition of dentin MMPs, with improved immediate bond strength, even if no change in bond degradation was detected after three months . Galardin (0.2 mM) can inhibit the proteolytic activity of demineralized dentin at concentrations approximately 10–100 times lower than that of CHX (2.2 mM) ( Fig. 4 ) .
3.1.3
Tetracycline
Tetracycline and their semi-synthetic analogues doxycycline and minocycline are a family of broad-spectrum antibiotics with cationic chelating proprieties that have been reported to serve as effective MMPs inhibitors . In addition to zinc chelation, they can down-regulate MMPs mRNA expression . Doxycycline, the only clinically available tetracycline with MMPs inhibiting properties, strongly decreases dentin matrix degradation . Broad-spectrum MMP inhibitors such as chemically modified tetracyclines (CMTs) that lack anti-microbial activity, but retain their MMP-inhibiting capacity (CMT-3, Metastat) are particularly effective in inhibiting MMPs in dentinal caries lesion . Their mechanism of action is thought to be by inhibition of the activity and secretion of the enzyme as well as calcium chelation . By binding to the enzyme’s active site zinc ion, CMTs can alter the conformation of the pro- enzyme molecule, thus blocking its catalytic activity in the extracellular matrix .
The effect of tetracyclines and their analogs on the preservation of resin–dentin bonds has not yet been investigated. Because of their MMP inhibiting potential, these compounds deserve further investigation. However the purple stain of teeth known to occur during photo-oxidation may render tetracycline-derived compounds not suitable for clinical use.
3.1.4
Biphosphonate
Another class of broad-spectrum proteases inhibitors, bisphosphonates, are known to be effective inhibitors of MMPs presumably by chelating the zinc and calcium ions from the enzymes . In a recent study, Tezvergil-Mulutuay et al. demonstrated potent inhibition of recombinant MMP-9 with less hydroxyproline release and loss of dry mass using polyvinylphosphonic acid (PVPA) . Similar to CHX, PVPA binds electrostatically to collagen. However, its use as a potential MMP inhibitor in dentin bonding may be more advantageous than CHX in that PVPA can be trapped in collagen matrices via the use of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) . These results suggest that the use of PVPA could enhance the longevity of the dentin–resin bonds. The literature in the field of bisphosphonates as MMP inhibitors is still scarce and thus future work should be performed to evaluate the long-term bond degradation of adhesive interfaces containing PVPA and bisphosphonates.
3.1.5
Quaternary ammonium compounds
Other cationic compounds, such as quaternary ammonium compounds, may also inhibit dentinal MMPs and thus stabilize the adhesive interface over time. Quaternary ammonium salts are positively charged at physiological pHs and have an effective antibacterial activity. Similar to CHX, these compounds are cationic, water-soluble, but unlike CHX they may not leach out of bonded interfaces. QAMs inhibit soluble MMP-9 as or more effectively than Galardin, and almost completely inhibit the demineralized dentin collagen degradation .
Polymerizable quaternary ammonium methacrylates (QAMs), especially 12-methacryloyloxydodecylpyridinium bromide (MDPB) have been incorporated into self-etching primers because they possess antimicrobial properties and can copolymerize with adhesive monomers . In vitro and clinical experiments have also indicated that QAMs (namely MDPB in Clearfil Protect Bond) may inhibit collagenolytic enzymes in the hybrid layer . MDPB proved to be the most effective quaternary ammonium methacrylate yet studied . However, other studies have reported reductions in bond strength comparable to other adhesives , so it may be too early to make any definitive conclusions of the clinical efficacy of MDPB in the preservation of hybrid layer. Antonucci et al. recently evaluated the synthesis of two dimethacrylates with quaternary ammonium functionalities , demonstrating the potential of these bioactive monomers. Whether blends containing quaternary ammonium groups can also exert anti-MMP properties at the hybrid layer and thus, increase the durability of resin–dentin bonds, requires further investigation.
Benzalkonium chloride (BAC) is a mixture of alkylbenzyl-dimethylammonium chlorides of various alkyl chains. It is a cationic surface-acting agent with a quaternary ammonium group used as antimicrobial agent and surfactant . BAC-containing etchants can be used with etch-and-rinse adhesives without affecting immediate bond strength to enamel or dentin . Tezvergil-Mutluay et al. demonstrated that 0.5% BAC concentrations completely inhibited soluble MMP-2, -8 or -9, and produced significant reduction in demineralized dentin collagen degradation. BAC molecule binds strongly to demineralized dentin even after rinsing increasing the amount that remains viable in the hybrid layer exerting its anti-MMP properties. The use of BAC as anti-proteolytic agent has gained attention only recently. The long-term survival of adhesive interfaces created with etch-and- rinse adhesive systems, which have been treated with various concentrations of BAC, has been investigated only recently with encouraging results .
3.2
Collagen cross-linking agents
Even though CHX inhibits both MMPs , and cysteine cathepsins , the potential disadvantage is that CHX may leach out of hybrid layers within 18–24 months . Cross-linking agents are considered an interesting option for improving the stability and resistance of collagen degradation within the demineralized dentin matrix . Covalent cross-linking agents produced with external cross-linkers are very stable, and may inactivate the active sites of dentin proteases by reducing the molecular mobility of the active site or by changing negatively charged ionized carboxyl groups into positively charged amides. Thus, an increase in the extent of cross-linking of the collagen fibrils prior to adhesive application may result in increased bonding durability.
This approach has been recently investigated by some authors with the aim to reinforce collagen fibrils through intermolecular cross-linking . Since lower biodegradation rates and high mechanical properties of collagen are desirable, the use of collagen cross-linking agents in adhesive procedures has gained increased popularity in recent years . The use of glutaraldehyde, proanthrocyanidin and genipin , riboflavin , as well as tannic acid and carbodiimide has been proposed to enhance the mechanical and structural stability of dentin collagen, leading to a stable dentin matrix network that, after resin infiltration, should provide a durable hybrid layer . Moreover, collagen cross-linking molecules have been reported to improve the resistance of uncross-linked or mildly cross-linked collagen matrices to degradation by bacterial collagenases , potentially contributing to the stabilization of the resin–dentin interface over time.
In addition to the proven efficacy of collagen cross-linking agents in chemical or physical modification of the dentin collagen substrate, the clinical applicability of these solutions is desirable. Even though the results so far have been promising, problems remain to be solved before the clinical applications of cross-linking products become available.
The mechanism of glutaraldehyde cross-linking is known to be dependent on the formation of covalent bonds between the amino groups of proteins and the aldehyde groups of glutaraldehyde. A recent study demonstrated an increased modulus of elasticity following treatment of acid-etched dentin with 5.0% glutaraldehyde for 1 min . By improving dentin’s mechanical properties, it is expected that glutaraldehyde would also contribute to improving the resistance to bond degradation. However, despite its efficacy , glutaraldehyde is strongly toxic.
Topical treatment with 3.75 mass% proanthocyanidin for 5, 15 or 30 s of 10-μm deep demineralized dentin provided enough cross-linking to protect dentin matrix when incubated in a 0.1 mass% solution of bacterial collagenase, while untreated controls lost all their demineralized dentin matrix after one hour of incubation in the collagenase solution .
Grape-seed extract is also effective and leads to an increase of the immediate dentin bond strength in reduced application times , but it stains the dentin brown and the durability of long-term bond strength remains to be examined.
Low-dose riboflavin has also been tested as dentin cross-linking agent in conjunction with UVA or dental blue light with good success . These studies have shown effective MMP inhibition and stabilization of the adhesive interfaces , as well as increased mechanical properties, stability, and resistance to dentin collagen degradation . Riboflavin displays additional advantages, which offer potential for application in adhesive dentistry, including its biocompatibility, easy application and activation with UVA blue light .
3.2.1
1-Ethyl-3-(3-dimethylamino-propyl) carbodiimide (EDC)
1-Ethyl-3-(3-dimethylamino-propyl) carbodiimide (EDC), a cross-linking agent with very low cytotoxicity, has shown promising results in reducing dentin collagen degradation and preserving dentin bond strength over time with clinically acceptable application times .
EDC contains a functional group with the formula RN C NR. The carbodiimide reacts with ionized carboxyl groups in proteins to form an O-acylisourea intermediate that can react with a non-proteinated amino group and an adjacent protein chain to form a stable covalent amide bond between the two proteins, with the only by-product being urea. This category of cross-linking agent may inactivate the active sites of dentin proteases by reducing the molecular mobility of the active site or by changing negatively charged ionized carboxyl groups into positively charged amides. Additionally, EDC can induce cross-linking of both helical and especially telopeptide domains in collagen and may also prevent telopeptidase activity that would normally remove bulky telopeptides from the specific peptide bond of collagenases . Increases in collagen stiffness may prevent MMPs from “unwinding” collagen peptides . Since this “unwinding” is necessary to allow MMPs catalytic site to cut the peptide , it would also effectively inhibit MMPs functional activity.
In a recent study Mazzoni et al. , demonstrated that the use of 0.3 M EDC pre-treatment improves durability and structural integrity of resin–dentin interfaces after 1 year when bonding procedure were applied with two different total-etch adhesives. In addition, the effect of 0.3 M EDC on dentinal MMPs activity by means of zymographic analysis was tested, demonstrating its efficacy to completely inactivate dentinal gelatinases.
In a further study authors showed nearly complete inactivation of MMP-2 and -9 when 0.3 M EDC pre-treatment was applied prior to bonding procedures with two different total-etch adhesives, while the application of the same two adhesives systems alone to acid-etched dentin resulted in activation of dentinal gelatinases. Moreover, as in situ zymography technique was performed with the aim to obtain precise localization of the MMP activity within the hybrid layer created by the tested total-etch adhesives without previous extraction of enzymes from the tissue. Gelatinolytic activity was clearly detectable within the hybrid layers and along the tubular wall dentin extending from the dentinal tubules. Furthermore, the location of the activity well correlates with the demineralized uninfiltrated collagen layer in simplified total-etch adhesives at the bottom of the hybrid layer, an area also known for nanoleakage expression and the presence of naked collagen fibrils , as well as with the effectiveness of a 0.3 M EDC primer applied before bonding to inactivate protease activity within the hybrid layer ( Fig. 5 ).
